@article{mbs:/content/journal/jgv/10.1099/vir.0.069716-0, author = "Biswas, Poulomi and Kundu, Anirban and Ghosh, Ananta Kumar", title = "Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases", journal= "Journal of General Virology", year = "2015", volume = "96", number = "1", pages = "95-105", doi = "https://doi.org/10.1099/vir.0.069716-0", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.069716-0", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", abstract = "Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5′ RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2′-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N 7-and ribose 2′-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5′ G*pppG end showed that guanine N 7 methylation occurred prior to the ribose 2′-O methylation to yield a m7GpppG/m7GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N 7- and 2′-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the K m values of N 7-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5′ end of viral RNA.", }